Certain chemical compositions exhibit luminescence. Such compositions emit light during exposure of the composition to externally applied light, or for a brief time after the exposure ends. The light emitted by the composition progressively decreases in intensity after the exposure to incoming light ends. Luminescent behavior of this nature includes the phenomena commonly referred to as "phosphorescence" or "fluorescence." It has long been known that significant information concerning the physical and chemical properties of a composition can be deduced from the luminescent behavior as by observing the rate of decay of the emitted light. However, real chemical compositions of interest normally exhibit a plurality of different decay rates which may entail decay times ranging from seconds to picoseconds. With very shortlived luminescence, it is normally not practical to obtain useful information about such compositions simply by exposing the composition to light, terminating the exposure and observing the decay of luminescence.
However, it is possible to obtain equivalent information by exposing the composition to excitation light having amplitude varying at a predetermined frequency and observing the luminescent response of the composition. Typically, the response includes a component varying in intensity at the same frequency as the excitation light. One characteristic of the emitted light which can be observed is the degree of modulation at the particular frequency used, i.e., the ratio between the intensity of the component at this frequency and the total intensity of the emitted light. Another characteristic of the emitted light which can be observed is the phase relationship between the cyclic variations in the emitted light and the variations in the excitation light, i.e., the degree to which variations in the emitted light lag behind variations in the excitation light. It has been the practice heretofore to conduct experiments of this nature at numerous frequencies and gather information such as degree of modulation, phase angle and the like at each such frequency. Utilizing known techniques, significant information concerning the physical and chemical characteristics of the composition can be deduced from the information gathered using plural frequencies.
Typically, the various frequencies of excitation light have been applied to the composition in sequence, one frequency at a time. Such sequential application of the various frequencies may be performed using an automated "scanning. Instrument arranged to apply the various frequencies in a predetermined sequence. Substantial time is required to collect data for all of the various frequencies. This approach is unsuitable for application in dynamic systems where the composition is changing with time. Accordingly, there have been attempts made to obtain similar information by applying light at various frequencies simultaneously and then measuring the response at all of these various frequencies simultaneously. In such simultaneous arrangements, the light emitted by the composition necessarily includes components at the various excitation frequencies employed, which components are intermingled with one another. It has been suggested that these components can be segregated from one another using a multichannel "parallel hardware" structure. In this approach, the instrument would necessarily include a number of signal channels at least equal to the number of frequencies to be monitored. Each such channel would include elements such as filters or the like arranged to limit the response of that particular signal channel to a particular one of the frequencies employed. This approach, however, suffers from serious disadvantages in that the instrument is necessarily limited to operation at only a few frequencies simultaneously. Accordingly, this approach has not been widely adopted.
Another multifrequency instrument is described in Bright et al., "A New Frequency-domain Fluorometer for the Rapid Determination of Picosecond Rotationalcorrelation Time", J. Applied Physics, Vol. 61, pp. 8-11 (January, 1987) and in Bright et al. "Rapid-scanning Frequencies-Domain Fluorometer with Picosecond Time Resolution, Applied Optics, Vol. 26, pp. 3526-3529 (1987). In this approach, a sample of the composition is excited with a stream of pulses from a laser operating at a predetermined pulse frequency. According to known principles of mathematics, excitation light varying in such a repetitive pulsatile fashion includes components varying at the fundamental or pulse frequency and also includes further components varying at integral multiples of the fundamental frequency, i.e., at harmonics of the pulse frequency. The light emitted from the sample will likewise include components at all of these frequencies. An additional modulation device may be used to further modulate the excitation light at additional frequencies and thus introduce additional frequencies into the emitted light. The emitted light is converted to an electrical response signal by a photo-multiplier tube, and the resulting electrical response signal is passed to a high frequency spectrum analyzer. The spectrum analyzer provides separate indications of the strength or modulation of the response signal at each frequency corresponding to one or another of the harmonic or additional frequencies. Although an instrument of this type is capable of acquiring data over a wide range of frequencies for a given sample in only about ten seconds or less, it suffers from a fundamental drawback in that it does not provide phase information at any of these frequencies.
Another approach is disclosed by Lakowicz et al., .Two-GHz Frequency-Domain Fluorometer," Rev. Sci. Instrum., Vol. 57, pp. 2499-2506 (October, 1986). The instrument disclosed by Lakowicz et al. also uses a pulsed laser to provide repetitive, pulsatile, excitation light incorporating many harmonics of the pulse frequency This instrument further incorporates a frequency synthesizer for generating a cross-correlation signal at a frequency equal to the frequency of a selected harmonic plus a small offset or delta frequency, typically 25 Hz. The light emitted from the sample is converted to an electrical response signal by a photodetector and the resulting electrical signal is mixed with the cross-correlation signal. This mixing yields an output signal at the same offset or delta frequency containing the phase and modulation information present in the component of the response signal at the selected harmonic A portion of a pulsatile excitation light is diverted prior to reaching the sample and directed to a photodetector to provide a reference signal, which is likewise mixed with the same cross-correlation frequency to provide a reference signal at the same offset or delta frequency. The phase angle for the selected harmonic is obtained by comparing the phasing of the reference and response signals at the offset or delta frequency, whereas the degree of modulation is obtained by comparing the modulation of the reference and response signals. This procedure is repeated using different cross-correlation signals, each at a frequency equal to a different selected harmonic plus the offset or delta frequency, so to collect a complete set of information incorporating phase angle and degree of demodulation at all different harmonics. Although this instrument exposes the sample to light modulated at many different frequencies simultaneously and provides a signal incorporating the response of the sample at all of the frequencies, the cross-correlation operation is still performed for only one such frequency at a time. Therefore, considerable time is consumed in a measurement. In this respect, the instrument disclosed by Lakowicz et al. suffers from the same drawback as the instrument applying individual excitation frequencies sequentially to the sample.
An abstract entitled "Direct Waveform Collection and Analysis of Phase Fluorometry Data" by Feddersen et al. appearing in the February, 1988 issue of BiophysicaI Journal (Vol. 53, No. 2, page 404a) proposes to digitize the cross-correlated response signal at the offset or delta frequency and likewise to digitize the excitation and reference signals at the offset or delta frequency in an instrument of the type proposed by Lakowicz et al. This is said to facilitate combination of all the data for all of the different harmonics in a global analysis "incorporating data at all of the various frequencies. The abstract states that such digitization "will prove to be very useful" in analysis of signals in some future "parallel fluorometer, where the digitized waveform will simultaneously contain all of the harmonic frequency content of the excitation source."
Accordingly, there have been unmet needs heretofore for still further improvements in instruments and techniques for monitoring the luminescent response of chemical compositions.